Notch receptors deliver signals that regulate cell fate decisions as well as other aspects of differentiation, proliferation and apoptosis for many different tissues and cell types. The active form of Notch, intracellular Notch (ICN), is generated by enzymatic cleavage events after interaction with Notch ligands on adjoining cells. ICN then translocates to the nucleus where it activates gene expression through interaction with the DNA binding protein, RBP-J? (RBPJ). Mutations that stabilize or result in ligand-independent generation of ICN are oncogenic in both mice and humans. Greater than 50% of T-acute lymphoblastic leukemia (T-ALL) in humans display oncogenic activation of Notch. Many mouse models exist that mirror this process, providing critical models with which to study mechanisms underlying both initiation and propagation of ICN-driven leukemia. Inhibitors that block Notch cleavage, ?-secretase inhibitors (GSIs), can suppress expression of ICN as well as proliferation of a subset of T-ALL cell lines, and therefore, have been tested in clinical trials. However, results of these trials have been disappointing due to limited anti-leukemic activity and toxicity issues. Therefore, understanding how oncogenic activation of Notch results in aggressive leukemia is of utmost importance to defining new targets for therapeutic intervention. Our preliminary data, using a novel cell line developed in our lab in which levels of Notch and RBPJ can be individually manipulated, suggest that GSIs do not mediate their growth inhibitory effects in T-ALL through abrogating RBPJ-dependent ICN-driven transcriptional activation. This suggests that ICN functions to promote leukemogenesis via a novel paradigm-challenging mechanism that does not require RBPJ. Therefore, the main focus of this proposal is to address the question: How does de-regulation of Notch lead to leukemia, and are there undefined mechanisms in play? We will explore both nuclear and extranuclear roles for ICN in maintaining proliferation of leukemia cells, as well as investigate the requirement for RBPJ in ICN-driven leukemogenesis initiated at multiple stages of T cell development in vivo. It is our hope that these studies will reveal new therapeutic targets for drug design or intervention for human leukemia.